CRM1-mediated Nuclear Export Is Required for 26 S Proteasome-dependent Degradation of the TRIP-Br2 Proto-oncoprotein

Jit Kong Cheong,Lakshman Gunaratnam,Stephen I-Hong Hsu

doi:10.1074/jbc.m708365200

Jit Kong Cheong, Lakshman Gunaratnam + Show 1 more

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https://doi.org/10.1074/jbc.m708365200

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Abstract

Overexpression of the proto-oncogene TRIP-Br2 (SERTAD2) has been shown to induce E2F activity and promote tumorigenesis, whereas ablation of TRIP-Br2 arrests cell proliferation. Timely degradation of many cell cycle regulators is fundamental to the maintenance of proper cell cycle progression. Here we report novel mechanism(s) that govern the tight regulation of TRIP-Br2 levels during cell cycle progression. TRIP-Br2 was observed to be a short-lived protein in which the expression level peaks at the G(1)/S boundary. TRIP-Br2 accumulated in cells treated with 26 S proteasome inhibitors. Co-immunoprecipitation studies revealed that TRIP-Br2 forms ubiquitin conjugates. In silico analysis identified a putative leucine-rich nuclear export signal (NES) motif that overlaps with the PHD-Bromo interaction domain in the acidic C-terminal transactivation domain (TAD) of TRIP-Br2. This NES motif is highly conserved in widely divergent species and in all TRIP-Br family members. TRIP-Br2 was shown to be stabilized in G(2)/M phase cells through nuclear entrapment, either by deletion of the acidic C-terminal TAD, which includes the NES motif, or by leptomycin B-mediated inhibition of the CRM1-dependent nuclear export machinery. Mutation of leucine residue 238 of this NES motif abolished the interaction between CRM1 and TRIP-Br2, as well as the nuclear export of TRIP-Br2 and its subsequent 26 S proteasome-dependent degradation. These data suggest that CRM1-mediated nuclear export may be required for the proper execution of ubiquitin-proteasome-dependent degradation of TRIP-Br2.

Highlights

The TRIP-Br/SERTAD ( referred to as TRIP-Br) family of mammalian transcriptional coregulators has been shown to play important roles in governing cell cycle progression, at least in part, by regulating the expression of E2F
Polyubiquitinated forms of TRIP-Br2-HA, GAL4DBD-hTRIP-Br2, and GAL4DBD-hTRIP-Br1 were visible only following treatment with MG132. ␤-Tubulin was used as a loading control in Western blot (WB) analyses of the whole cell extracts (WCE)
As our data indicated that CRM1-mediated nuclear export may be involved in maintaining a low steady-state level of TRIP-Br2 in G2/M phase cells and that Leptomycin B (LMB) stabilizes TRIPBr2 in these cells (Fig. 3I), we investigated whether the increased stability of the C-terminal TRIP-Br2-(1–179) truncated protein may be due to a loss of key nuclear export signal (NES) motifs

Summary

Introduction

The TRIP-Br/SERTAD ( referred to as TRIP-Br (transcriptional regulator interacting with the PHD-bromodomain)) family of mammalian transcriptional coregulators has been shown to play important roles in governing cell cycle progression, at least in part, by regulating the expression of E2F-. Deletion of the TRIP-Br2 C Terminus, Which Includes a Putative Nuclear Export Signal Motif, Inhibits TRIP-Br2 Degradation Independent of Its Ubiquitination Status—An analysis of GAL4DBD-hTRIP-Br2 truncation mutants (Fig. 5A, left panel) revealed that the C terminus of TRIP-Br2, which includes a PHD-Bromo interaction domain and an acidic TAD, is required for its protein turnover.

Results

Conclusion